Antibodies directed against binding associated epitopes

ABSTRACT

Binding of two members of a binding couple reveals epitopes which are revealed only after binding and the monoclonal antibody secreted from the hybridoma cell line CG-10 directed against these epitopes bind to the bound couple at a significantly higher affinity than their binding affinity to either of the two members themselves when not bound to one another.

RELATED APPLICATIONS

This is a division of application Ser. No. 09/433,420, filed Nov. 4,1999, now U.S. Pat. No. 6,143,876 allowed, which is a division ofapplication Ser. No. 09/235,592, filed Jan. 22, 1999, now U.S. Pat. No.6,020,468, issued Feb. 1, 2000, which is a continuation of applicationSer. No. 08/464,726, filed Jul. 31, 1995, now U.S. Pat. No. 5,925,741,issued Jul. 20, 1999, which is based upon PCT International ApplicationNo. PCT/US93/12639, filed Dec. 29, 1993, claiming priority of IsraeliApplication Nos. 104291 and 104767, filed Dec. 31, 1992 and Feb. 17,1993, respectively.

FIELD OF THE INVENTION

The present invention concerns novel antigenic epitopes which becomesubstantially more accessible after binding of two members of a bindingcouple, e.g. ligand-receptor binding, antibody-antigen binding, etc.These novel antigenic epitopes will be referred to herein at times asbinding associated epitopes (BAE). A specific aspect of the presentinvention concerns BAE which are revealed after virus-receptorinteraction, e.g. HIV-CD4 interaction.

The present invention further concerns antibodies, particularlymonocional antibodies, directed against BAEs and further concerns theuse of such antibodies or BAEs in diagnostics and treatment.

BACKGROUND OF THE INVENTION AND PRIOR ART

Binding of two members of a binding couple, e.g. a virus to its receptoron a cell membrane, is a complex interaction which may involve interalia, a conformational change in the receptor and likely also in theviral receptor-binding protein. The study or such conformational changesmay have various important therapeutic implications.

A virus-receptor interaction which has been studied extensively inrecent years is that of the HIV (Human Immunodeficiency Virus) to theCD4 protein which is expressed by and present on membranes of Tlymphocytes, some macrophages and likely also on several other kinds ofcells. An HIV protein, gp120, which has a binding affinity to the CD4receptor was discovered, and the receptor recognition sites in thisprotein have been at least partially identified. Seeing that the bindingbetween the HIV virus or its gp120 protein to the CD4 receptor and theoccurrences following such interaction are critical phases in theinfection process, it is believed that agents which will interfere withthese infection stages will likely be useful as drugs in treating AIDSand particularly in inhibiting the progress of the HIV infection. It hasbeen proposed to use antibodies which recognize either the CD4 receptor,the gp120 protein or the complex which is formed following binding, asit was believed that such antibodies may form useful agents ininhibiting the infection process. Monoclonal antibodies (mAbs) usefulfor this purpose have been proposed, amongst others, by Celada et al.1990 (J. Exp. Med., 172, 1143-1150), Celada 1992 (WO 92/05799) andHealey et aL. 1990 (J. Exp. Med., 172, 1223-1242). These referencesdisclosed antibodies directed against CD4 which were shown to preventsyncytium formation without interfering with the gp120/CD4 complexformation. However, all the antibodies described to date were not foundto be useful in treatment since they either bind well to CD4 receptorsand thus may interfere with the normal function of non-infected CD4bearing cells or, where the antibodies were directed to an epitope inthe virus and specifically in the gp120 protein, they were as a rulefound to be strain and even isolate-specific.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide antigenic epitopesassociated with binding of two members of a binding couple to oneanother (BAE).

It is another object of the present invention to provide bindingassociated antibodies capable of binding to a complex consisting of twomembers of a binding couple, with a higher affinity than to each memberby itself.

It is another object of the present invention to provide medicinal anddiagnostic uses of such epitopes or antibodies.

The remaining objects of the present invention will be revealed in thefollowing description and claims.

GENERAL DESCRIPTION OF THE INVENTION

The present invention is based on the surprising finding that uponbinding of two members of a binding couple, certain novel antigenicepitopes are revealed or exposed and as a result become accessible toantibodies. When a complex of the two members is injected to an animal,an immune reaction is elicited and some of the produced antibodies aresuch which bind to the complex with a substantially higher affinity thanto either of the two members individually.

Hybridomas producing such antibodies can be prepared and monoclonalantibodies produced by such hybnrdomas may be used for the isolation ofthe epitopes and for various diagnostic and therapeutic purposes.

The epitopes by themselves may be utilized for producing specificantibodies or in some cases for vaccination.

The novel epitopes of the invention may consist of an amino acidsequence present in one of the two members of the binding couple whichbecomes accessible to antibodies or resumes a new conformation afterbinding of the two members to one another; or may consist of a pluralityof sequences either all in one member or being distributed between thetwo members but become associated with one another to form an antigenicepitope, after binding of the two members to one another.

The present invention thus provides, by one of its aspects, an antigenicepitope which is a member of a group consisting of:

(i) an epitope consisting of an amino acid sequence in a member of abinding couple, which becomes substantially more accessible toantibodies or resumes a new conformation after binding of the twomembers to one another,

(ii) an epitope consisting of two or more amino acid sequences in amember of a binding couple which upon binding of the two members, becomeclosely associated to form an antigenic epitope, and

(iii) an epitope consisting of two or more amino acid sequences, atleast one being in one member of a binding couple, and at least oneother being in the other member of the binding couple and upon bindingof the two members, said two or more amino acid sequences become closelyassociated with one another to form an antigenic epitope;

said antigenic epitope being immunogenic.

An epitope of the kind defined under (i) will be referred to herein attimes as “linear revealed epitope”; an epitope of the kind defined under(ii) as a “discontinuous revealed epitope” and an epitope of the kinddefined under (iii) will be referred to herein at times as “combinationepitope”.

The novel BAE may be an epitope which is revealed or exposed in animmunocomplexed antigen, i.e. in an antibody-antigen complex; afterligand-receptor bindings, e.g. hormone-receptor,neurotrans-mitter-receptor, toxin-receptor, virus-receptor bindings;etc. A specific embodiment of the present invention concerns an epitopewhich is revealed after binding of a virus to its receptor, inparticular epitopes which are revealed or exposed after binding of HIVthrough its gp120 protein to a soluble or membrane associated CD4receptor protein. Another embodiment concerns an immunocomplexed gp120epitope, i.e. an epitope which is revealed or exposed after binding ofgp120 to an antibody against it produced in the body during an immunereaction following an HIV infection.

The present invention further provides, by another of its aspects,antibodies which bind to a complex consisting of two members of abinding couple with a substantially higher affinity than with each ofthe two members by themselves A specific embodiment of this aspect ofthe invention concerns antibodies which bind to a complex formed betweenthe HIV gp120 protein and the CD4 protein and such which bind to animmunocomplexed gp120 with a substantially higher affinity than toeither of the members of the complex by themselves.

A higher affinity of binding may be 5 fold, preferably 10 fold higheraffinity of binding to the complex as compared to binding affinity ofthe antibody to each of the members of the complex by themselves, astested by at least one standard assay such as ELISA, RIA(Radioimmunoassay), or by means of a FACS (Fluorescent Activated CellSorter) analysis. It should be noted that at times higher affinity ofbinding may be seen by such standard procedures, but may not be seen tothe same extent in other experimental procedures. For example, a crypticBAE normally effectively revealed after complex formation may also alsobe exposed in a protein without complex formation when for example,denatured on a SDS gel. In case the test is performed on proteins on anSDS gel, a higher affinity of binding to the complex may not be seen,although present in the non-denatured proteins.

The antibodies of the invention may be poly- or monocional, although forreasons of high specificity and ease of obtaining relatively largequantities, monoclonal antibodies are generally preferred.

The epitopes of the invention may be detected and isolated by variousmethods, some of which will be briefly detailed herein:

1. Western blotting analysis:

CD4 or gp120 are digested by a number of proteolytic enzymes. Theresulting proteolized fragments are gel electrophoresed on SDSacrylamide gels, and blots are prepared by transferring the separatedfragments from the gel to a suitable filter. The filter is then probedwith a series of labelled mAbs. A fragment that repeatedly binds to amAb of interest is transferred to a PVDF (poly venildifluoride-Millipore inc. Ma.) membrane and is then sequenced by any ofa number of sequencing methods known per se.

2. Pepscan analysis:

(see Geysen et al., 1984. Proc. Natl. Acad. Sci. U.S.A., 81: 3998-4002;Geysen et al., 1985. Proc. Natl. Acad. Sci., U.S.A., 82: 178-182). Aseries of synthetic peptides corresponding to the complete sequence ofthe CD4 or gp120 molecules are produced in multiwell ELISA plates bysolid phase Merrifield peptide synthesis. The synthetic peptides arethen screened by incubating the plates with labelled mAbs of interest.An antibody that binds to a specific peptide is therefore mapped to thatcorresponding sequence.

This method is suitable for linear revealed epitopes and is obviouslynot suitable for mapping discontinuous epitopes or combination epitopes.In order to identify discontinuous or combination epitopes, thefollowing method may be used.

3. Epitope Libraries:

(see Scott ct. al., 1990., Science 249: 386-390; Delvin et al., 1990.,Science 249: 404-406; Cwirla et al., 1990., Proc. Natl. Acad. Sci.U.S.A. 87: 6378-6382). A library consisting of a collection of theentire repertoire of combinations or peptide sequences presented on thesurface of filamentous phases is constructed. (Thus, for example, thecomplete collection of hexapeptides is 20⁶=6.4×10⁷ peptides). The phagecontaining an epitope of interest is then enriched by a Biopanningmethod in which a few microliters containing the entire library arefirst incubated with a suitable mAb in a flask. The library-mAb mixtureis then transferred to a petri dish containing immobilized streptavidin.Only phages bound by the biotinylated mAb will bind to the streptavidinin the dish and after washing away of the non-bound phages, the boundphage is grown in the plate and ultimately sequenced to reveal thedesired epitope.

The method under 3 is particularly suitable for the detection ofdiscontinuous epitopes or combination epitopes, due to the existence, insuch libraries, of “mimetopes”, i.e. linear peptides that functionallymimic such which can naturally be produced by discontinuous distantresidues.

In order to prepare the antibodies of the present invention laboratoryanimals are injected with complexes formed between the two members of abinding couple such as complexes formed between viruses or viralparticles and the receptor to which they bind, eg. gp120/CD4 complexesin the case of the HIV or with immunocomplexed viruses or viralparticles, e.g. immunocomplexed gp120. Following injection and thedevelopment of an immune reaction, spleen cells may be isolated fromthese laboratory animals and hybridomas may then be prepared by methodsgenerally known per se. The hybridomas are then screened for such whichsecrete antibodies which react with the complex with higher affinitythan with each of the individual components.

Hybridomas producing monoclonal antibodies of the invention constituteanother aspect of the present invention. One hybridoma cell linedesignated hereinbelow as CG-10 was deposited on Feb. 4, 1993, at theEuropean Collection of Animal Cell Culture (ECACC), Porton Down,Salisbury, Wiltshire, SP4 OJG, United Kingdom, and was assigned theAccession No. 93020415.

The antibodies of the invention can be used as a therapeutic agent for avariety of applications such as for the treatment of viral infections inorder to inhibit further propagation of the infection. For example,antibodies which are specific for gp120/CD4 complexes may have animportant potential use in the treatment of AIDS. As known, for humanmedicinal use, the mAbs should preferably be “humanized”, e.g. bymethods known per se such as CDR loop grafting (see Verhoyen et al.,1988, Science 239: 1534-1536). Alternatively, the mAbs should be ofhuman origin, i.e. human mAbs. Additionally, antibodies of the inventionmay have various diagnostic applications, e.g. anti-immunocomplexedgp120 be used for diagnosis or staging of HIV infections.

The epitope of the invention may have various uses such as indiagnostics, as well as in immunization. For similar uses alsoanti-idiotype antibodies against the above antibodies of the inventionmay be used. Such anti-idiotype antibodies also constitute an aspect ofthe invention.

In the following description specific reference will at times be made toHIV-related epitopes which become accessible to antibodies upon bindingof HIV or its gp120 protein to the CD4 protein and to ananti-immunocomplexed gp120 antibody and to antibodies which specificallybind to complexes formed between gp120 and CD4 proteins or bind toimmunocomplexed gp120. It will no doubt be appreciated by the artisanthat although these embodiments of the present invention are preferredembodiments, the invention is not limited thereto.

Antibodies available to date which were proposed for use in AIDStreatment were unsuitable for this purpose. On the one hand, prior artantibodies directed against different epitopes or gp120, were found tobe ineffective in inhibiting viral infections particularly in view ofthe very high strain and isolate variability of this protein. On theother hand, prior art antibodies directed against the CD4 protein mightinterfere with the normal functions of non-infected CD4-expressingcells. Against this, the antibodies of the present invention do notpossess these drawbacks associated with prior art antibodies. Theantibodies of the invention, in the specific case of HIV, arespecifically directed to epitopes which are revealed or become moreaccessible after interaction between the HIV and the CD4 protein, andwill thus inhibit progress of the infection, e.g. the formation ofsyncytia of lymphocytes, without interfering with the normal functionsof non-infected CD4-expressing cells. Furthermore, such antibodies arealso not likely to be strain or isolate specific, since even if theepitope is of a viral origin, the fact that it is not accessible priorto binding means that it is not subject to selective forces as in thecase of normally exposed epitopes. In addition, if the antibodies are ofa suitable kind, their binding to the complex may also elicit a cellularcytotoxic immune response against the infected cells.

The antibodies of the present invention may be conjugated to radioactiveor cytotoxic substances. Such conjugated antibodies will localize onlyon infected cells and will thus serve as specific targetedchemotherapeutic agents and will destroy only infected cells withoutseverely damaging normal, non-infected cells of the same kind.

Furthermore, anti immunocompiexed gp120 mAbs may serve in the diagnosisof HIV infections. To enable their use in detection in a diagnosticprocedure, such antibodies may preferably be conjugated to variousmarkers, such as radioactive or fluorescent substances, or enzymes suchas horseradish peroxidase etc.

By another aspect of the present invention, there is provided apharmaceutical composition for treating a viral infection comprising, asan active agent, an antibody according to the invention.

By another aspect of the invention there is provided an antiviralvaccine comprising as an active agent, an epitope of the invention.

By another aspect the present invention provides a method for treating aviral infection, comprising administering to a patient an effectiveamount of an antibody according to the invention.

By another aspect, the present invention provides a method for thediagnosis or staging of a viral infection, utilizing the antibodies ofthe invention. By one embodiment the method comprises contacting cellssusceptible of being infected by the virus with an antibody of theinvention, and then detecting the presence of such antibodies on thecells' surface. By another embodiment. The method comprises contacting abody fluid sample with an antibody against an immunocomplexed virus orviral particle or with such an antibody conjugated with a detectablemarker and then detecting the formation of immunocomplexes with saidantibody or conjugate. Where the antibodies of the present invention arenot conjugated to a detectable marker, a second antibody directedagainst the antibodies of the invention, conjugated to a detectablemarker will typically be introduced for the assay.

By another aspect of the invention there is provided a method forimmunizing an animal against a viral infection comprising administeringto a subject an effective amount of a BAE epitope of the invention, of akind which is revealed after binding of a virus to its receptor.

The invention will now be described with reference to specificembodiments described in the following examples, with reference at timesto the Figures in the annexed drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a series of scatchard plots obtained with five differentantibodies reacted with either the complex formed between gp120 and CD4or with either of these two agents by themselves: a) antibody CG-4; b)antibody CG-9; c) antibody CG-10; d) antibody CG-25; and e) antibodyCG-76.

FIG. 2 shows the affinity of binding of four of the antibodies to eitherthe complex or to CD4.

FIG. 3 is a micrograph showing syncytium formation between CD4expressing CEM cells and gp120 expressing BSC1 cells without antibodies(A) or in the presence of the various monoclonal antibodies (B-I).

EXAMPLES Example 1

Production of CD4/gp120 Complexes

Recombinant gp120 produced in a baculavirus expression system wasobtained from American Bio-Technologies. Recombinant soluble CD4,produced in Chinese hamster ovary (CHO) cells, was obtained from DuPont.CD4/gp120 complexes were prepared by a number of procedures. Forexample, 100 μg of gp120 can be mixed with 50 μg of CD4 in Tris bufferedsaline (TBS) at 4° C. for 12 hours. The complex is then dialysed againstthe same buffer prior to injection.

In order to verify that a CD4/gp120 complex was formed, ELISA assayswere used. In this assay, either gp120 or CD4 were immobilized on aplate and then a soluble counterpart (CD4 or gp120, respectively) wasadded to the plate. Subsequently, the plates were probed with a firstantibody being either anti-gp120 (obtained from AmericanBio-Technologies) or OKT4 and OKT4A (which are anti-CD4 antibodiesobtained from Orthodiagnostics Inc.) followed by a second alkalinephosphatase conjugated anti-mouse antibody.

For the ELISA assay Costar EIA/RIA 96 well plates (N3590) were coatedwith 50 μl of various concentrations of gp120 or CD4 in TBS(tris-buffered saline) overnight at 4° C. The plates were then washed inTBS and blocked with 3% BSA in TBS for 1 hour at room temperature (RT).Then 50 μl of 5 μg/ml solution of either recombinant CD4 or recombinantgp120 was added into the wells. The wells were rinsed and theappropriate mAb in 0.3% BSA/TBS was added to them and incubated at RTfor 2-3 hours. The wells were then washed with TBS and the secondantibody (alkaline phosphatase conjugated goat anti-mouse antibody[Sigma, A-0162]) was added (1:1000 in 0.3% BSA/TBS) and incubated for 1hour at RT. After washing the wells, they were reacted withp-nitrophenyl phosphate (1 mg/ml in 1M diethanolamine buffer pH 9.8/0.5mM MgCl₂) and read at 405 nm.

Exemplary results of such a complex analysis is shown in the followingTable I (the numbers in the Table represent OD×10⁻³):

TABLE I Plated Plated gp120 CD4 μg/ml OKT4A* OKT4 μg/ml OKT4A OKT4 10.027 640 5.0 206 729 5.0 23 773 2.5 135 492 2.5 27 313 1.25 42 291 1.25 29116 0.60 22 138 *OKT4A - an anti-CD4 antibody which recognizes only theCD4 protein which is not bound to gp120 (see: Sweet et al. (1991)Current Opinion in Biotechnology 2: 622-633).

Example 2

Production of Anti-complex mAbs

3 mice were immunized with a CD4/gp120 complex that had been extensivelydialyzed. A total volume of 1 ml complex prepared as in Example 1, wasdialyzed against 2 liters of Tris buffered saline (TBS), (total volume 6liters) for 12-14 hours at 4° C. These mice developed a good immuneresponse against both CD4 and gp120.

A plurality of hybridoma cell lines were prepared from these mice. Fromthe spleen of one of the injected mice, which was found to be extremelylarge, about 4×10⁸ cells were obtained and 4 aliquots of about 10⁸ cellswere taken separately. Each aliquot was fused with NS-1 cells. Twofusions were processed in parallel. A total of 1170 clones were obtainedand after 10 days of culture, the media were screened for antibodiesdirected against CD4/gp120 complex in an ELISA essay which was similarto that described above in Example 1 with a difference in that aCD4/gp120 complex was immobilized on the plates (5 μg gp120: 2.5 μgCD4/ml).

147 clones were found positive and these were then rescreened withimmobilized CD4/gp120 complex as well as separate immobilized CD4 andgp120 in a similar ELISA assay to the above. Of the original clones only81 continued to secrete antibodies and of these 15 were selected forfuture characterization. Out of these, 13 clones were found to be stableand were injected into mice for a successful production of ascitesfluids.

Ascites fluids of all 13 mAbs described were produced, lyophilized andanalyzed for their ability to bind CD4, gp120 and CD4/gp120 complexes.The mAbs were tested by ELISA assays, FACS analysis and Western Blots. Acollection of 10 mAbs was identified as being interesting for furtheranalysis and the result of their binding studies is shown in thefollowing Table II.

TABLE II mAb subclass gp120/CD4 gp120 CD4 1 CG-1 IgG1 +++ − +/− 2 CG-4IgG1 +++ +++ − 3 CG-7 IgG1 +++ − +/− 4 CG-8 IgG1 +++ − +/− 5 CG-9 IgG1+++ − ++ 6 CG-10 IgG1 +++ − − 7 CG-25 IgG1 +++ − ++ 8 CG-30 IgG1 +++ −++ 9 CG-40 IgG1 +++ +++ − 10  CG-76 IgG1 +++ − +++ In the above table,+++ shows a strong reaction whereas a − shows no reaction.

Of these antibodies, several including those designated CG-1, CG-7,CG-8, CG-9, CG-10, CG-25 and CG-30, are such having the characteristicsof the antibodies of the present invention. Of those antibodies, CG-10,as can be seen from the above results, is specific only for theCD4/gp120 complex.

Example 3

Scatchard Analysis of Five mAbs.

A series of scatchard analyses were performed in order to determine thebinding affinity of five mAbs (CG-4, CG-9, CG-10, CG-25 and CG-76) forthe CD4/gp120 complexes as compared to their binding affinity forisolated CD4 or gp120.

CD4, gp120 and CD4/gp120 complexes were immobilized in wells of ELISAplates. The tested mAbs were iodinated with ¹²⁵I and the immobilizedantigens were incubated with the iodinated mabs. The scatchard plots ofthe five above mentioned mAbs are seen in FIG. 1. The affinity ofbinding of four of the mAbs to either the complex or to the CD4 is shownin FIG. 2.

mAbs, CG4 and CG-76 showed similar binding affinities to isolated CD4and CD4/gp120 complexes and may therefore be of the type previouslyreported by Celada et al.

As opposed to this mAbs CG-9 and CG-25 showed a respective 10 and 100fold higher binding affinity to the CD4/gp120 complex, as compared totheir binding affinity to CD4 alone. Furthermore, mAb CG-10 had noaffinity for the isolated CD4 and bound exclusively to theCD4/p120complex.

Example 4

Inhibition of Syncytium Formation

The formation of syncytia between vaccinia BSC1 cells (African greenmonkey kidney cells) infected with the recombinant vaccinia clone VEP16(see Ashorn et al., 1990 J. Virol. 64 2149-2156) expressing gp120 ontheir surface and CEM cells (a human T helper lymphocyte cell line)expressing CD4 on their surface was tested. Generally, BSC1 cultureswere infected with recombinant vaccinia (5 pfu/ml) expressing cellsurface gp120. These cells were then mixed with CEM cells in thepresence of varying amounts of mAbs and incubated for different periodsof time. The degree of syncytia formation was monitored and thus theextent of neutralization potential for the various mAbs was estimated.

As is shown in panel A of FIG. 3, when infected BSC1 cells were mixedwith CEM cells, syncytia were formed within a few hours. The potentialof the different antibodies to neutralize syncytium formation was testedby pre-incubation of CEM cells for 5 to 12 hours with the tested mAbsbefore the addition of the infected BSC1 cells (panels C, G, H and I) orby the addition of the tested mAbs simultaneously with the BSC1 cells(panels B, D, E and F).

The results are shown for the following mAbs which were added in theindicated amounts:

Panel B: 10 μg of mAb CG-9;

Panel C: 1μ or mAb, CG-25;

Panel D & G: 1 μg of CG-76;

Panel H: of 1μ mAb CG-10; and

Panel I: 1 μg of CG-76.

As seen in FIG. 3, all the tested mAbs (CG-9, CG-10, CG-25 and CG-76)showed at least some syncytium neutralizing activity, each to adifferent extent. In addition, the pre-incubation of the mAbs with CD4expressing CEM cells improved the neutralizing activity of the mAbs.

Example 5

Preliminary Analysis of Sera Obtained from HIV+ Hemophilia Patients

Sera from five HIV+ hemophilia patients were tested for their potentialto inhibit the binding of several mAbs to CD4/gp120 complexes in acompetitive ELISA.

For the competitive ELISA assay Costar EIA/RIA 96 well plates (N3590)were coated with 50 μl of 5 μg/ml CD4/gp120 complex in TBS(tris-buffered saline) overnight at 4° C. The plates were then washed inTBS and blocked with 3% BSA in TBS for 1 hour at room temperature (RT).Then, the wells were rinsed and 50 μl of the appropriately diluted mAbin 0.3% BSA/TBS was added to them with 50 μl of TBS (control wells) or50 μl of the mAb and 50 μl of the tested serum were added simultaneously(test wells) and incubated at RT for 2-3 hours. The wells were thenwashed with TBS and the second antibody (alkaline phosphatase conjugatedgoat anti-mouse antibody [Sigma, A-0162]) was added (1:1000 in 0.3%BSA/TBS) and incubated for 1 hour at RT. After washing the wells, theywere reacted with p-nitrophenyl phosphate (1 mg/ml in 1M diethanolaminebuffer pH 9.8/0.5 mM MgCl₂) and read at 405 nm.

The results of the competitive ELISA are shown in Table 3.

TABLE III #mAb Pat. #1 Pat. #5 Pat. #8 Pat. #9 Pat. #11 CG-1 50 40 55 700 CG-4 78 87 68 75 60  CG-7 59 61 68 70 0 CG-8 43 60 58 71 0 CG-9 11 3820 27 0 CG-10 65 87 98 91 68  CG-25 25 44 22 41 0 CG-30  0 40  0 21 0CG-40 16 44  0  8 0

The numbers (N) in the above table represent the % of inhibitioncalculated as follows: $N = {\left\lbrack {1 - \frac{\begin{matrix}{{O.\quad D.\quad {of}}\quad {binding}\quad {of}\quad {mAbx}\quad {to}\quad {{CD4}/{gp120}}} \\{{in}\quad {the}\quad {presence}\quad {of}\quad {the}\quad {tested}\quad {serum}}\end{matrix}}{{O.\quad D.\quad {of}}\quad {binding}\quad {of}\quad {mAbx}\quad {to}\quad {{CD4}/{gp120}}}} \right\rbrack \times 100}$

Thus, for example, serum from patient no. 11 inhibited 68% of thebinding of mAb CG-10 to immobilized complex.

The results in Table III show that sera from all five tested hemophiliapatients can compete with the binding of mAb-CG-10 to the CD4/gp120complex. It appears, therefore, that HIV infected hemophiliacs containcomplex specific antibodies and that mAbs of the present invention maythus serve in the diagnosis of HIV infections.

Example 6

Detecting the Presence of gp120 in a Blood Sample Using the CG-10 mAb

The close proximity assay is a method for measuring the existence ofreceptorligand binding without the need to separate bound from freeligand (Hart H. E. and Greenwald, E. B. Mol Immunol. 16, 265-267 (1979);Udenfriend. S. et al. PNAS 82, 8672-8676 (1982); Udenfriend S. et al.Anal Biochem. 161 494-500 (1987); U.S. Pat. No. 4,568,649). This assaymakes use of beads that have been impregnated with a scintillating dyehaving a desired receptor immobilized on their surface. The beads aremixed with a radioactive ligand and while the radiation emitted bynon-bound ligand molecules in solution is quenched by the medium, theemission of a ligand which is bound by the receptor coating thescintillating beads, due to the close physical proximity, can interactwith the dye and elicit a secondary photon. This scintillation is easilymonitored and quantitated in a standard scintillation counter (in theabsence of any additional scintillating fluid).

CD4 is immobilized on the surface of commercially availablescintillating beads (Amersham product #RPN 141) using a commerciallyavailable mAb such as OKT4 or other mAbs such as CG-76 (see Example 2).

Alternatively, beads impregnated with a fluorophor (obtained fromNuclear Enterprises, Scotland, product #NE-102A) are directly coatedwith CD4 as follows: methyl groups are oxidized to COOH groups eitherwith dilute nitric acid or with KMnO₄ and these are used to couple theCD4 via the water soluble carbodiimide reaction. The beads are washed toseparate excess carbodiimide and unreacted CD4.

A tested blood sample is solubilized with Triton-X-100 and cleared fromerythrocytes by methods known per se. The beads coated with CD4 are thenmixed with the blood sample and a radioactive labeled CG-10 mAb (or anyother mAb directed against the CD4/gp120 complex) is added to themixture. The sample is read in a standard scintillation counter; a highreading indicating the presence of gp120 in the tested blood sample.

Another application in which CG-10 could be used as a diagnostic couldemploy such methods as Immuno-PCR in which the DNA template would beconjugated to CG-10 immunoglobulin. This could be done with theglycomoiety of the antibody.

Example 7

Production of Anti gp120/V3 Loop Complex mAbs

A gp120/anti-HIV-1V3 loop complex was prepared by mixing a mAb directedagainst anti-HIV-1 V3 loop_(IIIB) designated M77 (obtained from AdvancedBioscience Laboratories, Inc., MD USA-ABL) with recombinant HIV-1gp120_(IIIB) (obtained from American Bio-Technologies-ABT). BALB/c micewere immunized with the prepared complex and hybridoma cell lines wereprepared from the injected mice. Media from several clones obtained fromthese hybridomas were screened for antibodies directed against thegp120/anti V3 loop complex in an ELISA assay as described in Example 2.The clones that were found positive were retested for their affinity forthe above complex as well as for separate gp120 and M77 mAb. mAbs thatbound gp120_(IIIB) were also tested for their affinity of bindingHIV-2_(ST) (a preparation of recombinant gp120 derived from anHIV-2_(ST) isolate, purchased from SmithKline Beecham, King of Prussia,Pa.). The results of the binding studies of several of the mAbs is shownin the following table 4.

TABLE IV Clone Type Complex M77 120_(IIIB) 120_(ST) 3F lgG1 1.204 0.0350.193 — 4D lgG1 1.093 0.044 1.090 0.629 4G lgG2b 0.840 0.033 0.907 0.6864H lgG1 1.063 0.083 0.379 0.564 7A lgM 0.884 0.045 0.944 1.238 7F lgG2a1.136 0.092 0.214 — 8G lgG1 1.149 0.064 0.141 —

Of these antibodies, those designated 3F and 8G, are such having thecharacteristics of the antibodies of the present invention, i.e.specific for the gp120/anti V3 loop complex and bind at very lowaffinity to the M77 mAb and gp120_(IIIB).

What is claimed is:
 1. A method for immunizing an animal against a viralinfection comprising administering to a subject an effective amount ofan epitope or an anti-idiotype antibody of said epitope, wherein saidepitope is of a complex formed between two members of a binding coupleand is a member of a group consisting of: (i) an epitope consisting of asequence in a member of a binding couple, which becomes substantiallymore accessible to antibodies or resumes a new conformation afterbinding of the two members to one another, (ii) an epitope consisting oftwo or more sequences in a member of binding couple which upon bindingof the two members, become closely associated to form an antigenicepitope, and (iii) an epitope consisting of two or more sequences, atleast one being in one member of a binding couple, and at least oneother being in the other member of the binding couple and upon bindingof the two members, said two or more amino acid sequences become closelyassociated with one another to form an antigenic epitope; said antigenicepitope being immunogenic.
 2. The method of claim 1, wherein the epitopeis revealed after antibody-antigen or ligand receptor binding.
 3. Themethod of claim 1, wherein the epitope is revealed after virus or viralparticle-receptor binding.
 4. The method of claim 1, wherein the epitopeis revealed after HIV or gp120-CD4 binding.
 5. The method of claim 1,wherein the epitope binds to a monoclonal antibody produced by the GC-10hybridoma deposited with the European Collection of Animal Cell Culture(ECACC) under the accession No.
 93020415. 6. The method of claim 5,wherein the epitope is the anti-idiotype of a monoclonal antibodyproduced by the GC-10 hybridoma.
 7. The method of claim 4, wherein theepitope is a sequence in the gp120 protein.
 8. The method of claim 2,wherein the epitope is revealed after binding of gp120 to an anti-gp120antibody.
 9. The method of claim 8, wherein the epitope is a sequence inthe gp120 protein.